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Structures and interaction analyses of integrin ?M?2 cytoplasmic tails.

ABSTRACT: Integrins are heterodimeric (? and ? subunits) signal transducer proteins involved in cell adhesions and migrations. The cytosolic tails of integrins are essential for transmitting bidirectional signaling and also implicated in maintaining the resting states of the receptors. In addition, cytosolic tails of integrins often undergo post-translation modifications like phosphorylation. However, the consequences of phosphorylation on the structures and interactions are not clear. The leukocyte-specific integrin ?M?2 is essential for myeloid cell adhesion, phagocytosis, and degranulation. In this work, we determined solution structures of the myristoylated cytosolic tail of ?M and a Ser phosphorylated variant in dodecylphosphocholine micelles by NMR spectroscopy. Furthermore, the interactions between non-phosphorylated and phosphorylated ?M tails with ?2 tail were investigated by NMR and fluorescence resonance energy transfer (FRET). The three-dimensional structures of the 24-residue cytosolic tail of ?M or phosphorylated ?M are characterized by an N-terminal amphipathic helix and a loop at the C terminus. The residues at the loop are involved in packing interactions with the hydrophobic face of the helix. 15N-1H heteronuclear single quantum coherence experiments identified residues of ?M and ?2 tails that may be involved in the formation of a tail-tail heterocomplex. We further examined interactions between myristoylated ?2 tail in dodecylphosphocholine micelles with dansylated ?M tail peptides by FRET. These studies revealed enhanced interactions between ?M or phosphorylated ?M tails with ?2 tail with Kd values ?5.2±0.6 and ?4.4±0.7 ?m, respectively. Docked structures of tail-tail complexes delineated that the ?M/?2 interface at the cytosolic region could be sustained by a network of polar interactions, ionic interactions, and/or hydrogen bonds.

SUBMITTER: Chua GL

PROVIDER: S-EPMC3243538 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Structures and interaction analyses of integrin αMβ2 cytoplasmic tails.

Chua Geok-Lin GL Tang Xiao-Yan XY Amalraj Monalisa M Tan Suet-Mien SM Bhattacharjya Surajit S

The Journal of biological chemistry 20111103 51

Integrins are heterodimeric (α and β subunits) signal transducer proteins involved in cell adhesions and migrations. The cytosolic tails of integrins are essential for transmitting bidirectional signaling and also implicated in maintaining the resting states of the receptors. In addition, cytosolic tails of integrins often undergo post-translation modifications like phosphorylation. However, the consequences of phosphorylation on the structures and interactions are not clear. The leukocyte-speci ...[more]

PMID: 22052909

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Project description:Integrin activation, the rapid conversion of integrin adhesion receptors from low to high affinity, occurs in response to intracellular signals that act on the short cytoplasmic tails of integrin beta subunits. Talin binding to integrin beta tails provides one key activation signal, but additional factors are likely to cooperate with talin to regulate integrin activation. The integrin beta tail-binding proteins kindlin-2 and kindlin-3 were recently identified as integrin co-activators. Here we report an analysis of kindlin-1 and kindlin-2 interactions with beta1 and beta3 integrin tails and describe the effect of kindlin expression on integrin activation. We demonstrate a direct interaction of kindlin-1 and -2 with recombinant integrin beta tails in pulldown binding assays. Our mutational analysis shows that the second conserved NXXY motif (Tyr(795)), a preceding threonine-containing region (Thr(788) and Thr(789)) of the integrin beta1A tail, and a conserved tryptophan in the F3 subdomain of the kindlin FERM domain (kindlin-1 Trp(612) and kindlin-2 Trp(615)) are required for direct kindlin-integrin interactions. Similar interactions were observed for integrin beta3 tails. Using fluorescence-activated cell sorting we further show that transient expression of kindlin-1 or -2 in Chinese hamster ovary cells inhibits the activation of endogenous alpha5beta1 or stably expressed alphaIIbbeta3 integrins. This inhibition is not dependent on direct kindlin-integrin interactions because mutant kindlins exhibiting impaired integrin binding activity effectively inhibit integrin activation. Consistent with previous reports, we find that when co-expressed with the talin head, kindlin-1 or -2 can activate alphaIIbbeta3. This effect is dependent on an intact integrin-binding site in kindlin. Notably however, even when co-expressed with activating levels of talin head, neither kindlin-1 or -2 can cooperate with talin to activate beta1 integrins; instead they strongly inhibit talin-mediated activation. We suggest that kindlins are adaptor proteins that regulate integrin activation, that kindlin expression levels determine their effects, and that kindlins may exert integrin-specific effects.

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Structures and interaction analyses of integrin ?M?2 cytoplasmic tails.

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Structures and interaction analyses of integrin αMβ2 cytoplasmic tails.

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